The intestinal epithelium has a remarkable capacity to repair and regenerate after injury; however, our understanding of underlying mechanisms is incomplete. We recently reported that following intestinal epithelial injury by the chemotherapeutic, doxorubicin, intestinal stem cell (ISC) numbers increase during crypt regeneration. Importantly, concomitant with ISC expansion, we demonstrated dramatic expansion of the Paneth cell compartment associated with increases in both Paneth cell number and size. Paneth cells reside at the base of small intestinal crypts, and intercalated among them are ISC. This places Paneth cells in an advantageous position to influence ISCs, and impact ISC survival, proliferation or cell fate decisions. The precise role of Paneth cells in creating or maintaining the ISC niche, especially during injury and repair is not defined. Our central hypothesis is that crypt and ISC injury induces a change in Paneth cell function and secretory products which promote expansion and survival of intestinal stem cells and Paneth cells during epithelial repair. To begin to test this hypothesis we propose the following Specific Aims: 1) to use a newly developed cryptidin2-tomato Paneth cell reporter mouse to directly visualize Paneth cell responses to injury in vivo, 2) to isolate and study functional characteristics of Paneth cell from normal and regenerating epithelium, and 3) to compare the secretory products of Paneth cells isolated from normal, damaged, and regenerating intestinal epithelium. These studies are significant because they will define cellular and molecular mechanisms linked to epithelial repair after intestinal damage by a clinically used therapeutic agent. Defining Paneth-cell derived trophic or pro-repair factors will point to potential therapeutic directions to alleviate adverse effects of chemotherapies on intestine. The studies are innovative because they use a novel Paneth cell reporter model, novel stem cell culture models and extend upon new findings that Paneth cell expansion is an integral component of epithelial repair after injury. PUBLIC HEALTH RELEVANCE: Paneth cells have been viewed primarily as part of the intestinal mucosal innate immune system protecting against microbial infection and overgrowth. The studies detailed in this proposal are designed to determine if Paneth cells play an additional role in promoting intestinal stem cell growth and repair following injury. This would be a novel role for these cells, and defining Paneth-cell derived trophic or pro-repair factors will point to potentially novel therapeutic directions to alleviate adverse effects of chemotherapies on intestine.